Project Summary Much of our genome is made up of interspersed repeats derived from the activities of mobile genetic elements. There are subsets of these sequences that become activated in cancers. Our research lab and others have shown that nearly half of cancers fail to restrain long interspersed element-1 (LINE-1, L1) sequences. L1 is an active retrotransposon that codes for two proteins, an RNA binding protein (ORF1p) and a protein with endonuclease and reverse transcriptase activities (ORF2p). Paradoxically, though these proteins are pervasively expressed in many human cancers, they inhibit cell growth in culture. We have exciting new data from genome-wide knockout screens demonstrating how tumor suppressor gene mutations found in cancers enable them to survive and grow despite their expression of L1. More importantly, these screens have also identified genes that become essential in cells coping with L1 expression. These synthetic lethal genes represent unique molecular vulnerabilities for L1(+) cells. L1(+) cells require specific DNA repair pathways, effective replication stress signaling pathways, and replication fork restart capabilities. Here, we will test the hypothesis that these pathways are required to eliminate L1 insertion intermediates and prevent collisions between these intermediates and DNA replication forks. This project will increase our understanding of how cancer cells replicate their DNA. It could lay a foundation to leverage L1-associated DNA replication stress to limit cancer cell growth.